From the high speed supernatant of Novikoff hepatoma cells, eight factors which are capable of increasing the rate and extent of DNA synthesis by the homogeneous Novikoff beta-polymerase have been identified. Four of these proteins have been highly purified. Most are relatively inactive with heterologous polymerases and with homologous alpha- and Mt-polymerases. Factor IId is active with Novikoff alpha- and guinea pig beta-polymerases, while factor IV is also active with homogeneous guinea pig beta-polymerase. All factors are low or absent in normal liver except factor I which is higher in liver than the hepatoma. The proposed research is aimed at continuing studies of mammalian DNA polymerase accessory proteins by 1) identifying any factors participating in or regulating DNA replication/repair; 2) purifying each to homogeneity; 3) characterizing each with regard to enzyme activity (if any), MW, subunit structure, stoichiometry of its action on in vitro DNA synthesis, binding to DNA and/or DNA polymerase, subcellular localization and amount present under different physiological conditions; 4) clearly establish a role for each in DNA replication and/or repair; 5) reconstruct DNA synthesis complexes in vitro using defined natural DNA templates, polymerase and factors. Purification will follow established methods for protein purification. The criterion for homogeneity will be a single band in polyacrylamide gels and localization of activity to that band by assaying sliced, eluted sister gels. Each factor will be assayed for exo- and endonuclease, RNA polymerase, DNA polymerase, DNA ligase, RNase, ATPase and DNA unwinding activity. Binding to DNA or DNA polymerase will be determined by gel filtration. Subcellular localization and factor level quantitation will be determined by fluorescence and radioimmunoassay techniques, respectively.